The increasing prevalence of drug resistant HIV compromises the continued utility of current HIV drugs. Virtually all clinically used drugs are directed at only two HIV targets, protease and reverse transcriptase (RT), thus one approach to the problem of HIV drug resistance is to develop therapeutics directed at novel HIV targets, since therapeutics directed at novel HIV targets will almost certainly be active against current drug- resistant virus strains. One such target is RT-associated ribonuclease H (RNaseH), an essential enzyme activity for HIV replication and the only HIV enzyme not yet targeted by any clinically used or pipeline therapeutics. We have identified acylhydrazones as interesting compounds in that certain analogs are potent inhibitors of both HIV-1 RT DNA polymerase and RT-RNaseH activities. We hypothesize that this bifunctional inhibition is due to binding of the inhibitor to two distinct sites on RT, one of which is in or near the RNaseH domain. We recently obtained a crystal structure of acylhydrazones in the RT polymerase (pol) domain, so detailed understanding of the interaction of acylhydrazones with the RT RNaseH domain will be invaluable for design of both new RNaseH inhibitors (RNaseHI) and new bifunctional inhibitors. To this end, we propose four Specific Aims, (1) to determine the structure of HIV-RT RNaseH in complex with acylhydrazone inhibitors. Detailed NMR studies of the interaction of acylhydrazones with an active isolated HIV-1 RT RNaseH domain will provide precise structural information of the inhibitor binding pocket in the RNaseH domain, the microscopic conformation of RNaseH-inhibitor interaction sites, and influence of inhibitor binding on RT-RNaseH structure;(2) to validate the RNaseHI binding pocket determined in Aim 1. The RNaseHI binding pocket determined in Aim 1 will be validated in the context of intact RT and HIV-1 virus by evaluating the effect of mutation of selected residues interacting with RNaseHI, in the RNaseH fragment (biochemical and NMR structural analyses), in intact RT, and in HIV-1;(3) to optimize the inhibitory potency of acylhydrazone and analog inhibitors. Several approaches will be used to identify and develop potent monofunctional (RNaseH-specific) and bifunctional inhibitors, including screening of a library of 5000 hydrazone derivatives and synthesis of new analogs based on structures of acylhydrazones in the RNaseH and pol site binding pockets;(4) to conduct detailed biochemical and virologic characterizations of new RNaseHI. The inhibitory properties of potent RNaseHI and bifunctional acylhydrazones from Aim 3 will be characterized with purified RT and in cell-based HIV replication studies (including resistance development). We hypothesize that bifunctional inhibitors may be preferable in the context of resistance development. Selected RNaseHI will be further evaluated in NMR structural studies to better define the binding pocket for RNaseHI. This structural information along with the crystal data will provide a firm basis for rational design of new inhibitors. PUBLIC HEALTH RELEVANCE: There are more than 20 drugs for the treatment of HIV infection, yet the virus continues to spread in the US and worldwide. Furthermore, drug resistant HIV is increasing, limiting treatment options for individuals infected with these strains. New drugs are essential. This project will explore compounds directed at a novel HIV target, ribonuclease H, with the goal of developing new drugs that will be active against existing drug resistant HIV.